Mitochondria are central to the intrinsic apoptotic pathway of programmed cell death. Commitment to cell death occurs upon mitochondrial outer membrane permeabilization (MOMP), which results in the release of cytochrome c (Cyt c) into the cytoplasm, triggering caspases and irreversibly leading to cell death MOMP is regulated by the complex interplay of cell signals and the BCL-2 family of proteins. Dysregulation of this vital apoptotic control point is implicated in various pathologies, including cancer, diabetes, autoimmune conditions, and others, making this checkpoint a promising target for pharmacological intervention.
Accordingly, a means of precisely monitoring and quantifying the MOMP process is desirable. Such a tool would enable the assessment of treatments which act upon BCL-2 and other apoptosis-modulating factors. Currently, MOMP is monitored by optical techniques. Several fluorescence probes are sensitive to mitochondrial membrane potential, such as TMRE, JC-1 and rhodamine 123. Additionally, various immunoassays can ascertain Cyt c concentrations after release. While extremely useful, the prior art tools suffer from various shortcomings. First, the installation of the fluorescent probes into the relevant compartments of the mitochondria can create artifacts not present in intact mitochondria. Secondly, the number of cells required for a detectable signal in optical assays of MOMP is high, for example, with some assays requiring at least 3 or more micrograms of mitochondrial protein.
Accordingly, there is an ongoing need in the art for tools which allow precise monitoring and quantification of the MOMP process without associated artifacts and which are enabled using small samples. Provided herein are novel sensors and associated methods of using such sensors, which sensors and methods provide the art with a means for the facile, sensitive, and accurate assessment of MOMP. Advantageously, the sensors of the invention are highly scalable and enable massively parallel screening of agents for activity which promotes or inhibits the intrinsic apoptotic pathway.